Hematopoietic stem cell (HSC) gene therapy (GT) holds promise for curing diseases for which there is currently inadequate or highly toxic treatment. There remains an unmet need to develop effective HSC-GT that achieves a greater level of engraftment with reduced conditioning regimen toxicity. We propose to use the dog model of HSC-GT to study key problems that can translate into significant advances for treatment of hematological and other diseases. The aims of this proposal are designed to improve the efficacy and safety of autologous HSC-GT with (1) increased engraftment by infusion of greater numbers of ex vivo expanded gene modified (GM)-HSC, (2) reduced toxicity by greatly reducing chemo/radiotherapy conditioning prior to GM-HSC infusion. The studies are designed to achieve results that will be translated into improving human HSC-GT. Aim 1. Increase the cell dose of autologous GM-HSC with a 2-week ex vivo expansion of CD34+ cells. Identify the optimal combination of small molecules/drugs during the ex vivo expansion of GM-HSC. For each recipient dog, compare two expansion methods in an in vivo competitive repopulation assay. One half of the CD34+ cells will be marked with green fluorescent protein (GFP) expressing lentiviral vector (LV) and the other half with mCherry (mC) expressing LV. After 9.2 Gray total body irradiation (TBI) and GM-HSC infusion, measure the hematopoietic reconstitution of green vs. red cells. This will identify the drug combination and expansion method that achieves optimal sustained engraftment of GM-HSC. To assess the clonal diversity of the progeny of individual HSCs after transplantation, each transduced cell will have a unique molecular barcode LV. Use high throughput sequencing to track GM-HSC progeny after transplantation long term. Aim 2. Reduce the cytotoxic conditioning regimen needed for long term engraftment of GM-HSC. To increase the competitive repopulating advantage of ex vivo expanded GM-HSC; administer the CXCR4 antagonist plerixafor (AMD3100) to increase the unoccupied HSC niches in the bone marrow just prior to low-dose TBI followed by infusion of GM-HSC. Next, test if KIT (CD117)-specific tyrosine kinase inhibitor (TKI) can increase engraftment of GM-HSC. We will test the hypothesis that the GM-HSCs have a competitive advantage over the plerixafor or TKI-treated endogenous irradiated HSC. If successful, we would combine plerixafor + TKI to assess if this non-cytotoxic regimen could achieve GM-HSC engraftment with less TBI. Plerixafor + TKI followed by GM-HSC infusion may be repeated to further increase engraftment of GM-HSC. Finally, we will test the optimal GM-HSC transplant regimen to correct the erythroid disease in pyruvate kinase (PK) deficiency dogs with the R-type PK gene to achieve a functional cure of anemia. Upon completion of these aims, we will have defined highly translatable approaches to increase engraftment of GM-HSC, reduce conditioning regimen toxicity and induce immune tolerance to GM-HSC.